Protein kinase C phosphorylation of the EGF receptor at a threonine residue close to the cytoplasmic face of the plasma membrane

Abstract

The receptor for epidermal growth factor (EGF) is a 170,000-180,000 molecular weight single-chain glycoprotein of 1,186 amino acids. Its sequence suggests that it has an external EGF-binding domain, formed by the NH2-terminal 621 amino acids, linked to a cytoplasmic region by a single membrane-spanning segment. In the cytoplasmic portion, starting 50 residues from the membrane, there is a 250-residue stretch similar to the catalytic domain of the src gene family of retroviral tyrosine protein kinases, and, indeed, a tyrosine-specific protein kinase activity intrinsic to the receptor is stimulated when EGF is bound. Increased tyrosine phosphorylation of cellular proteins, detected in A431 cells following EGF binding, may be important in the mitogenic signal pathway. Tumour promoters such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA), counteract this increase, as well as causing loss of a high affinity class of EGF binding sites. The major receptor for TPA has been identified as the serine/threonine-specific Ca2+/phospholipid-dependent diacylglycerol-activated protein kinase, protein kinase C. By substituting for diacylglycerol, TPA stimulates protein kinase C. Protein kinase C phosphorylates purified EGF receptor at specific sites, and this reduces EGF-stimulated tyrosine protein kinase activity. TPA treatment of A431 cells increases serine and threonine phosphorylation of the EGF receptor at the same sites, which suggests that the reduction of EGF receptor kinase activity in TPA-treated cells is a consequence of the receptor's phosphorylation by the kinase. We have attempted to identify these phosphorylation sites and show here that protein kinase C phosphorylates threonine 654 in the human EGF receptor. This threonine is in a very basic sequence nine residues from the cytoplasmic face of the plasma membrane in the region before the protein kinase domain; it is thus in a position to modulate signalling between this internal domain and the external EGF-binding domain.

Prolactin modulates phosphorylation, signaling and trafficking of epidermal growth factor receptor in human T47D breast cancer cells

“…Two major threonine phosphorylation sites are known in the EGFR juxtamembrane cytoplasmic domain, Thr654 and Thr669 (Hunter et al, 1984;Davis and Czech, 1985;Heisermann and Gill, 1988;Takishima et al, 1988). PKC may directly mediate the Thr654 phosphorylation, whereas Thr669 is thought to be phosphorylated by ERKs (Northwood et al, 1991;Takishima et al, 1991).…”

Retinoids arrest breast cancer cell proliferation: retinoic acid selectively reduces the duration of receptor tyrosine kinase signaling

“…In vitro and in vivo PKC phosphorylates serine and threonine residues in the juxtamembrane region of the EGFR, a region involved in ligand-dependent internalization and receptor down-regulation. One clear PKC site is threonine 654 [72], although the role of PKC phosphorylation of threonine 654 per se in affecting the numbers of high-affinity surface receptors and EGF signaling is unclear [69,[73][74][75][76][77]. One possible explanation for retinoid inhibition of EGFR signaling is a PKCα-dependent phosphorylation of the EGFR in the juxtamembrane region.…”

Inhibition of stimulus‐dependent epidermal growth factor receptor and transforming growth factor‐α mRNA accumulation by the protein kinase C inhibitor staurosporine

“…The present study describes the ability of staurosporine to block certain cellular responses in the MDA468 breast cancer thought to be mediated by protein kinase C. The activation of protein kinase C by TPA in the MDA468 cells, as in many others, results in the loss of high-affinity EGFbinding sites [19,20]. This effect of TPA is believed to be mediated through phosphorylation of the EGF receptor at Thr 654 by protein kinase C [21][22][23].…”